The connections between neurons compose the basic structure of the brain, through which all of its functions are expressed. Mapping and characterizing these connections is fundamental to neuroscience, as no brain system can be understood if its architecture is unknown. Neuroanatomical tracers have been used for decades to reveal connectivity in much of the brain, but the information they provide is limited. Tracers are compounds which are transported along axons and allow their origin, destination, or both to be visualized. It is difficult to control the specificity of existing tracers, which leads to inconsistent and unreliable results. To accurately map connectivity, the existence of synaptic connections between identified neurons must be verified at the electron microscopy (EM) level. Furthermore, EM studies of synapse morphology should ideally be carried out on labeled connections. The few existing tracers that are compatible with EM are not compatible with morphological studies due to severely compromised ultrastructure. We have conducted extensive morphological and neuroanatomical tracer studies at the EM level on our system of interest, the adult rat lateral amygdala. We propose to develop novel tracers which will label specific cells for light microscopy and EM while preserving high quality ultrastructure for morphological studies. Using a viral vector, we will express a membrane-targeted form of the EM label horseradish peroxidase (HRP) in adult rat neurons. Unlike current tracers, HRP can be visualized without subjecting tissue to detergents which damage EM ultrastructure. This allows good preservation of tissue morphology, while restricting the HRP to the membrane prevents the label from obscuring any cellular organelles. We will place the membrane-bound HRP gene under the control of one of two different promoters. The first, the calcium/calmodulin-dependent protein kinase II promoter, will restrict expression of the label to excitatory neurons. Combined with the fact that viral transfection is restricted to cell bodies (which conventional tracer uptake is not), this will be the most spatially and functionally specific tracer available. The second promoter will be from the activity-regulated cytoskeleton-associated protein Arc, which is expressed in response to strong synaptic activation and behavioral experience. This will allow identification of the axons and dendrites of cells activated during learning and plasticity experiments such that their synapses can be specifically examined. The tools we propose to create will have a broad range of applications in neuroanatomy, neurobiology, and plasticity studies throughout the brain. PUBLIC HEALTH RELEVANCE: Mapping and characterizing the connectivity between brain cells is fundamental to understanding how the brain works. Many neurological diseases involve dysfunction of particular brain circuits or connections, and it is essential to elucidate and examine these connections in both normal and diseased brains. The goal of this project is to develop novel tools to map neural connections and visualize them clearly at the microscopic level.